Immune response is a regulatory mechanism indispensable against infection for a living body, and immune cells are patrolling constantly in the living body, to respond rapidly to various sources of infection. Such characteristics that constitutive cells are moving continuously are not recognized in other complex living systems, and have been developed specifically in the immune system. Among the immune cells, cells such as neutrophils, macrophages are known to function during primary defense of infection, while T- and B-lymphocytes trigger antigen-specific immune response by recognizing external foreign substances via the antigen receptor. The above T- and B-lymphocytes differentiate in primary lymphoid organs such as thymus and bone marrow, and transfer to a particular compartment in second lymphoid organs such as spleen, lymph nodes, Payer's patch (lymphoid organs in the small intestine), and by recognizing antigens gathered there from various organs via the antigen receptor, induce specific immune response. At that time, the transfer of lymphocytes to a particular site of second lymphoid organ is very important for the formation of immune response. Heretofore, the transfer of the lymphocytes was known to be induced by protein called generally various chemokines, while the molecule mechanism that controls the mobility of the lymphocytes themselves remained unknown.
Change of cell polarization and cytoskeletal reorganization were indispensable for the cells movement (Cell 84, 359-369, 1996), and these were known to be controlled by G protein of low molecular weight such as Rho, Rac and Cdc42 (Proc. Natl. Acad. Sci. USA 92, 5027-5031, 1995; Science 279, 509-514, 1998; J. Cell Biol. 141, 1147-1157, 1998; Science 287, 1037-1040, 2000). Among these, Rac particularly provides driving force at the time of cell migration, by forming an actin-rich protrusion, called foliar protrusion (Science 279, 509-514, 1998; Cell 103, 227-238, 2000). On the other hand, molecules showing structural homology called CED5, DOCK 180 and Myoblast city (MBC) were identified in Caenorhabditis elegans, human and Drosophila melanogaster. These molecules are called CDM family molecules by their initials, and all of them are thought to be related to cytoskeletal reorganization by functioning upstream of Rac (Cell 84, 359-369, 1996; J. Cell Biol. 138, 589-603, 1997; Nature 392, 501-504, 1998; Genes Dev. 12, 3331-3336, 1998; Genes Dev. 12, 3337-3342, 1998; Nature Cell Biol. 2, 131-136, 2000). Although genetic analysis with the use of a deletion mutant has shown that the above CED-5 and Myoblast City are crucial for cell migration of particular types of cells, (J. Cell Biol. 138, 589-603, 1997; Nature 392, 501-504, 1998; Nature Cell Biol. 2, 131-136, 2000), physiological relevance of the CDM family proteins in mammals remained unknown.
It is known that DOCK2 (KIAA0209; DNA Res. 3, 321-329) encodes another member of the CDM family proteins, which is specifically expressed in human haematopoietic cells, and that the DOCK2 binds to activate Rac in 293T kidney cells (Biochem. Biophys. Acta 1452, 179-187, 1999). On the other hand, the present inventors isolated a new gene Hch belonging to the CDM family from mouse thymus cDNA library, and found that the gene product comprises 1828 amino acids, and encodes SH3 domain at the N terminus (Nature, 412, 826-831, 2001). Moreover, the present inventors confirmed by Northen Blot analysis using mouse organs that whereas DOCK180 was expressed in various organs, the expression of Hch was restricted to thymus and spleen. Further, by an analysis using cell lines they confirmed that Hch expression was observerd in all T-, B- and macrophage cells, with the exception of two mutant T-cell lines. Furthermore, it has been revealed that a significant change in cell morphology and enhancement of adhesion were observed by introducing Hch into mutant T-cell line lacking Hch expression. Though 1677 of the 1828 amino acids encoded by Hch are identical to human DOCK2, and Hch was thought to be mouse DOCK2 homologue, the physiological function remained unknown.
The present inventors identified DOCK2 as a molecule belonging to the CDM family, expressing specifically in lymphocytes as mentioned above, and by generating the knockout mice, they revealed that the molecule was indispensable to lymphocyte migration (Nature, 412, 826-831, 2001). In DOCK2-deleted lymphocytes, active Rac is not detected by any of chemokine stimulation. Therefore, it can be thought that DOCK2 regulates lymphocyte migration via Rac activation. However, it remains unknown by which mechanism DOCK2 activates Rac. Rac functions as a molecule switch, and is activated by a GDP/GTP exchange factor (GEF). Though DOCK2 binds with Rac, it is hard from its structure, to think that it functions as GEF. Therefore, it is estimated that DOCK2 activates Rac by recruiting GEF via other molecules.
Recently, CED-12 being a molecule that associates with CED-5, which is one of the CDM family molecules, and that regulates cytoskeleton has been identified in C. elegans, and ELMO-1, -2 and -3 were reported as their mammalian homologues (Cell, 107, 27-41, 2001). Moreover, several dozens of GDP/GTP exchange factors (GEF) were known heretofore, and among these GEFs, as a molecule functioning as Rac-specific GEF, the following are known: Tiam-1 and -2 that determines the invasion to thymoma cell lines (Cell, 77, 537-549, 1994; Nature, 375, 338-340, 1995); Vav1 that regulates T cell receptor signal (Nature, 385, 169-172, 1997) besides Vav2, Vav3; Trio (J. Cell Science, 113, 729-739, 2000); STEF (J. Biol. Chem., 277, 2860-2868, 2002); and P-Rex1 (Cell, 108, 809-821, 2002). All these five molecules have a common domain, and comprise a function to provide GTP to Rac.
Autoimmune diseases and graft rejection are caused by the invasion of lymphocytes into the target organ. Therefore, it is thought that DOCK2 might be a suitable target molecule to treat or prevent such diseases or pathology. The object of the present invention is to identify the functional domain of DOCK2 by using a deletion mutant, to screen a substance interfering in the binding of DOCK2 and SH3 domain of DOCK2, particularly to provide a method for screening a substance interfering in the association of DOCK2 and ELMO, a method for screening a substance interfering in the association of ELMO and GDP/GTP exchange factor such as Tiam, or a method for searching therapeutic agents for immune-related diseases, such as allergy, autoimmune diseases, GvH or graft rejection with the use of these screening methods, and the like.
DOCK2 is a molecule expressed specifically in lymphocytes, comprised of 1828 amino acid residues including SH3 domain, that activates Rac and regulates cytoskeleton to determine lymphocyte mobility. The present inventors have made a keen study to solve the above object, found that Rac-activating ability was significantly decreased in DOCK2 mutant lacking 504 amino acid residues in the N terminus including SH3 domain of DOCK 2, and that actin polymerization could not be induced, and they identified ELMO1 as a molecule binding to this domain. Moreover, as the binding of DOCK2 and ELMO1 was completely inhibited by the single amino acid mutation of SH3 domain, they have found that DOCK2 associates with ELMO1 via SH3 domain. Furthermore, they have found that ELMO1 binds with Tiam1 functioning as Rac-specific GDP/GTP exchange factor (GEF). In other words, they have found that DOCK2 activates Rac by recruiting Tiam1 via ELMO1. Therefore, they found that by inhibiting intermolecular interaction of SH3 domain of DOCK2, ELMO1 and Tiam1, the artificial control of lymphocyte migration was possible. The present invention has been thus completed with this knowledge.